(1) Clinical applications arising from stem cell and regeneration research will require an understanding not only of de novo tissue synthesis but also of the patterning mechanisms regulating blastema morphology. This project seeks to elucidate the regulatory networks controlled by biophysical epigenetic changes which orient blastema polarity relative to existing tissues. Our hypothesis is that ion transport by the H,K ATPase transporter and gap junctional communication (GJC) together regulate anterior-posterior polarity during planarian regeneration upstream of traditional signaling. Aim 1 is to identify and characterize transcriptional targets downstream of ion transport, through microarray analyses of regenerating worms inhibited for H,K ATPase (completed) and GJC activity. Candidates will be selected using specific criteria, validated by expression analyses, and functionally characterized following RNAi knockdown using in situ hybridization and molecular marker analyses of phenotypes. Aim 2 is to build and test models which outline the mechanisms ion transporters use to control axial identity in regenerating tissue. A preliminary transcriptional network downstream of ion transport will be generated by identifying network nodes, using quantitative PCR to test how RNAi knockdown of a single candidate affects the expression of all other candidates. Hypotheses predicted by this network will then be generated and tested, to elucidate the transcriptional and signaling response to the activity of the implicated ion transporters during the establishment of regenerative polarity. The long-term goal is to compile and validate a systems-level regeneration network model integrating biophysical and biochemical signaling. This has the potential for use as a predictive model of morphogenesis to aid in the translation of basic regeneration research into practical biomedical therapies. (2) The promise of stem cell and regeneration research is ultimately to replace lost or damaged tissues, organs and limbs in patients suffering from injury, aging or cancer. Although the regeneration of new tissue is critical, for that tissue to be functional, it must be properly patterned in relationship to the existing tissues: This research aims to discover how new and old tissues communicate to regulate this pattering, by learning how regenerating flatworms determine whether to form a new head and/or tail following amputation.